1. Field of the Invention
This invention relates to a polypeptide found to be present in the venom of the Agelenopsis aperta spider and to polypeptides having substantially the same amino acid sequence and substantially the same activity as said polypeptide. The polypeptides and the pharmaceutically-acceptable salts thereof block calcium channels in cells including neuronal and muscle cells of various organisms including invertebrates and vertebrates. This invention also relates to the use of said polypeptides and their salts in blocking calcium channels in cells such as cells in the nervous and muscular system of an organism, per se; in the treatment of calcium channel mediated diseases and conditions in a mammal; and in the control of invertebrate pests. Further, this invention relates to compositions comprising said polypeptides and salts thereof.
2. General Background
It has been reported that the venom of the spider Agelenopsis aperta contains at least two toxins which affect calcium currents. Jackson, H., et al., Soc. Neu. Sci. Abstr. 12:1078 (1987). Those authors disclose a toxin, referred to therein as AG2, which has a molecular weight of less than 1,000 daltons and appears to suppress calcium currents in a broad range of tissues. Further, Jackson, H., et al., Soc. Neu. Sci. Abstr. 12:730 (1986) report another toxin from Agelenopsis aperta comprising a component of about 6,000 M.W. That toxin is reported to effect presynaptic blockade of transmission and it has been suggested that the toxin blocks calcium channels associated with the release of neurotransmitter.
Certain polypeptides found to be present in the venom of the Agelenopsis aperta spider are disclosed in U.S. patent application Ser. No. 07/346,181, filed Apr. 28, 1989, now abandoned. Those polypeptides are disclosed therein as blockers of calcium channels in cells and are described as follows according to fractions in which they were found:
Fraction G:
An amino-terminal amino acid sequence comprising: ##STR1##
Fraction H.sub.1 : ##STR2##
Fraction H.sub.2 : ##STR3##
Fraction I: ##STR4##
Fraction J:
An amino-terminal amino acid sequence comprising: ##STR5##
Fraction K: ##STR6##
Fraction L.sub.1 :
An amino-terminal amino acid sequence comprising: ##STR7##
Fraction L.sub.2 :
A polypeptide having the following identifying characteristics:
(a) present in a fraction from crude venom of the Agelenopsis aperta spider which elutes off a C-18 Vydac.RTM.22 mm .times.250 mm, 300 .ANG. pore size, 10 .mu. particle size column using a flow rate of 15 ml/min., a solvent system using a linear gradient program of 5% .fwdarw.20% B, 95% .fwdarw.80% A [0 .fwdarw.30 min.]then 20% .fwdarw.70% B, 80% .fwdarw.30% A [30 .fwdarw.55 min.], where A is 0.1% aqueous TFA and B is acetonitrile, at about 43 minutes; and
(b) present in a fraction of the fraction described in (a), above, which elutes off a C-18 Vydac.RTM.10 mm .times.250 mm, 300 .ANG. A pore size, 5 .mu. particle size column using a flow rate of 3.5 ml/min. and a solvent system using a linear gradient program of 25% .fwdarw.40% B, 75% .fwdarw.60% A [0 .fwdarw.30 min.], where A is 0.1% aqueous TFA and B is acetonitrile, at about 22.5 minutes.
Fraction M:
An amino-terminal amino acid sequence comprising: ##STR8##
Compounds which are calcium antagonists have a variety of utilities. Calcium antagonists can find clinical application in the treatment of such conditions as angina, hypertension, cardiomyopathies, supraventricular arrhythmias, aesophogeal achalasia, premature labor and Raynaud's disease among others. See W. G. Nayler, Calcium Antagonists, Academic Press, Harcourt Brace Jovanovich Publishers, New York, NY 1988, the teachings of which are incorporated herein by reference. Further, such compounds are useful in the study of the physiology of cells such as neuronal and muscle cells and in the control of invertebrate pests.